Recovery of Salmonella bacterial isolates from pooled fecal samples from horses

Abstract Background It is important to determine if a horse is shedding Salmonella spp., but a complete culture series can be cost prohibitive. Objectives Determine the optimal pooling technique to maintain high sensitivity of Salmonella spp. culture using spiked samples, and then demonstrate the efficacy of this protocol on clinical submissions. Hypothesis Pooled fecal samples are as sensitive as 5 individual cultures for the detection of Salmonella shedding. Animals A single Salmonella‐negative horse from the university herd, and 19 hospitalized horses. Methods Salmonella‐free fecal samples were spiked with different amounts of Salmonella spp. (102, 103, 104, and 105 colony forming units [cfu]) and homogenized to evaluate pooled samples. Five individual fecal samples were collected from 19 hospitalized horses. Ten‐gram aliquots of each individual sample were combined to make a pooled sample. Both individual and pooled samples were cultured for Salmonella spp. The identity of bacterial isolates was confirmed by matrix‐assisted laser desorption‐ionization time of flight mass spectrometry. Results A 102 cfu concentration of Salmonella spp. could be recovered from a spiked Salmonella‐free fecal sample. Homogenization protocols indicated that the addition of 20 mL of broth to the pooled sample improved recovery, whereas homogenization time did not. Of the 19 horses tested, 5 were positive for Salmonella. In all instances, Salmonella spp. were recovered from the fecal pool as well as individual samples. Conclusions and Clinical Importance Pooling of 5 fecal samples for Salmonella culture is a sensitive and cost‐effective diagnostic approach to detect horses that are shedding the organism.


| INTRODUCTION
It is important to identify horses that are shedding Salmonella spp. in their feces because of zoonotic risks and biosecurity concerns. Doing so can be difficult because horses with Salmonella spp. infections are often asymptomatic or can be clinically indistinguishable from those with colitis caused by other pathogens. Additionally, horses may shed the organism intermittently. Previous studies 1,2 found that a single culture identified between 33% and 55% of horses that were later found to be positive when additional cultures were performed. Why Salmonella and other enteric pathogens are shed intermittently is not well understood. It is hypothesized to be the result of local immune and inflammatory factors, but the exact mechanisms have yet to be elucidated. 3 Because of the intermittent nature of fecal shedding, the most widely accepted protocol for diagnosing Salmonella in horses is collecting 5 individual fecal samples for Salmonella culture. 2 As the cost of laboratory testing increases, however, submitting 5 individual cultures has become cost prohibitive in many instances. This expense leads practitioners to submit fewer fecal cultures.
Pooled fecal cultures are a well-established cost-effective way to estimate the prevalence of Salmonella infection in cattle and swine herds. 4,5 They generally are performed by combining feces collected on a single day from multiple animals. Pooling samples has been described as a means to identify horses with Salmonella using PCR but not culture. 6 Although PCR has an important role in the identification of Salmonella positive animals, PCR results do not always match fecal culture results. 7 An animal that is intermittently shedding the organism might be misidentified as a nonshedder by a single PCR just as it can be misidentified by a single culture. Although PCR assays are improving in their agreement with bacterial cultures, PCR is still not as reliable as culture. 8 Additionally, isolates obtained by culture are required for serotype determination.
Our goal was to determine if pooling 5 fecal samples collected at individual time points was as accurate as culturing 5 single samples to identify feces that contain Salmonella spp.

| MATERIALS AND METHODS
The study protocol was approved by Purdue University's Institutional Animal Care and Use Committee. Spiking and pooling of feces: Salmonella was cultured in broth and the cfu/mL were determined by standard plate counts. Desired infective doses of 10 2 , 10 3 , 10 4 , and 10 5 total cfu were obtained by serial dilution. These concentrations were selected because they correspond to what is frequently obtained in food animal pooled samples. 4 Feces in 20-g aliquots from the fecal donor horse were spiked with each desired concentration. Spiked samples were mixed using an electric homogenizer (Stomacher 400Circulator [Seward, UK]) for 1 minute at 230 rpm. Ten grams were cultured for Salmonella to show spiking was successful.
In phase 1 of the pooling study, the remaining 10 g were pooled with 40 g of Salmonella-negative feces, replicating a pool of 1 positive field sample and 4 negative field samples. Fecal pools were homogenized for 1 minute at 230 rpm.
In phase 2 of the pooling study, 4 homogenization methods were tested on triplicate 50 g pools containing a single 10 g sample spiked with 10 2 total cfu of Salmonella to determine the ideal homogenization protocol. Each pool then was divided for 5 individual cultures.
The homogenization protocols tested were: 1 minute with no broth, 5 minutes with no broth, 1 minute with 20 mL broth, and 5 minutes with 20 mL broth. All pools were processed at 230 rpm. The broth used in the homogenization protocol was Nutrient Broth (BBL, BD Life Sciences). It is formulated with peptone and beef extract and serves as a general-purpose broth (Table 1). Study population: Horses included in the study were presented to the teaching hospital between September 2018 and April 2021. Study eligibility was restricted to those horses for which clinicians elected to submit 5 individual samples for Salmonella culture either as a screen or because they were classified as high risk. Horses that exhibited clinical signs consistent with gastrointestinal disease such as diarrhea or colic, or horses with fever or leukopenia were cultured most frequently. Signalment, presenting complaint, days spent in the hospital, age, sex, breed, outcome, and culture results (individual samples and pooled sample) were obtained from the horse's medical record (see Table S1).   Table S1. The median age was 8 years (range, 1-21 years). The median number of days of hospitalization was 9 days (range, 2-160 days). The most common presenting complaint was colic (n = 8, 42.1%), followed by diarrhea (n = 4, 21%), colitis (n = 2, 10.5%) and fever (n = 2, 10.5%). Two of the 5 horses presented with both diarrhea and fever.
Horses with ophthalmic disorders (n = 2; 10.5%) were sampled because they shared barns or caretakers with horses that tested positive for Salmonella spp. at some point during their hospitalization. One horse was euthanized during hospitalization. The remainder were discharged from the hospital (n = 18, 94.7%).  Table S2. In summary, we found that pooling 10-g aliquots of 5 fecal samples and then culturing the pooled sample was as likely to detect a horse shedding Salmonella as submitting 5 individual samples. Because doing so decreased the number of samples submitted to the laboratory by 4, it represents a substantial cost savings. A sensitive and cost-effective clinical strategy for detecting Salmonella in feces from horses would be to submit a pool of 5 fecal samples for Salmonella culture.

Funding provided by Purdue University College of Veterinary
Medicine-Equine Research Advisory Board.

CONFLICT OF INTEREST DECLARATION
Authors declare no conflict of interest.

OFF-LABEL ANTIMICROBIAL DECLARATION
Authors declare no off-label use of antimicrobials.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION
Approved by Purdue University IACUC.

HUMAN ETHICS APPROVAL DECLARATION
Authors declare human ethics approval was not needed for this study.